1. Field of the present invention
The field of the present invention is the automatic conversion of the anilinothiazolino derivatives (ATZ) to the phenylthiohydantoin derivatives (PTH) of peptides and proteins in the well-known Edman degradation process.
2. Description of Prior Art
The principles of the solid phase Edman degradation technique for high sensitivity analysis of peptides and proteins are well-known. Briefly, the process can be described as follows. First, the protein or peptide is covalently bonded to an inert support, such as a polystyrene or glass support. While in a reaction column or vessel, this peptide or protein undergoes a coupling step using phenyl isothiocyanate (PITC) which may be in the radioactive form when labelling is desired. A buffer solution is pumped simultaneously with the PITC, and preferably for a period of time thereafter to clean the lines. After the coupling step, the phenylthiocarbamyl peptide or protein is alternately washed with methanol and with dichloroethane. The sample then undergoes a cleavage step by being treated with anhydrous trifluoroacetic acid. After the cleavage step, the protein or peptide derivatives in the reaction column are preferably washed with methanol. The resulting product after this cleavage step is an anilinothiazolinone (ATZ) amino acid derivative, which is usually unstable and normally requires a conversion step to a phenylthiohydantoin (PTH) amino acid derivative. These PTH derivatives are then identified by conventional means of chromatography.
The first two of these steps (viz. coupling and cleavage) have been automated, and incorporated into automatic "sequencers" (see U.S. Ser. No. 803,689 entitled "Microsequencing System for the Sequencing of Peptides and Proteins" by A. Bonner and M. Horn and references cited therein). These sequencers have gained general acceptance by offering many advantages over manual methods, among these being the time- and labor- saving aspects of automation, and the improved per-cycle yields, allowing a larger number of repetitive degradative steps to be performed on a peptide or protein sample.
The automation of the conversion step, however, has not kept pace with the automation of the other two steps. Several prototype conversion devices have been constructed for particular applications. One such known device is described by B. Wittmann-Liebold et al., in Analytical Biochemistry, Vol. 75, pp. 621-633 (1976), and in the German Offenlegungsschrift No. 2,208,088.
Several factors influenced the design considerations of the prior art. Thus, since the automated main sequencers of the prior art were already provided with programmers, controls, and supplies for nitrogen and vacuum, and since these relatively expensive components were also needed for automatic conversion, the trend of the industry was uniformly toward incorporation of the conversion apparatus into the main sequencer in order to avoid duplication. Incorporation of the conversion devices in the main sequencers, however, brought about various disadvantages. For example, major modifications were required in the electronics of the main sequencer, and since there are many different types of main sequencers, the prior art modifications for the conversion devices were therefore useful only in the particular sequencer into which they were incorporated. In addition, as a result of total incorporation into the main sequencer, the prior art conversion devices were synchronously tied to the main sequencer in such a way that neither the main sequencer nor the conversion component had independent versatility of operation or control. An additional drawback of the integrated systems of the prior art was that the operation of the conversion device also required simultaneous operation of the main sequencer, and as a result, at the end of the last conversion step, there would always remain in the reaction cell of the main sequencer one cycle's worth of unconverted sample. This resulted in loss of material and inefficient use of sequencing reagents, a matter of particular significance when the quantities of sample available are extremely small, and when the reagents are extremely costly.